A long-term goal of this project is to better understand the molecular basis of B lymphocyte development and immunoglobulin (Ig) gene rearrangement and expression. Two genes, lambda5 and VpreB, are expressed specifically in B cell precursors but not in B cells, and their gene products are associated with the mu H (heavy) chain on the pre-B cell surface. The function of these molecules is, at present, unknown. In this project we propose to test the hypotheses that lambda5 and VpreB are involved in a) pre-B cell adherence to or stimulation by stromal cells, b) induction of kappa light chain gene rearrangement, and/or c) inhibition of further H chain variable to diversity, joining (V to DJ) gene rearrangement. The normal cell populations which express lambda5 and VpreB will be identified and the molecular mechanisms which regulate the cell- stage specificity of their expression will be determined. Studies to directly test the above hypotheses will be performed by modulating the expression of these genes in early precursor or differentiating pro- and pre-B cell lines. This project will develop pro- and pre-B cell lines by IL-3 culture and Abelson murine leukemia virus (A-MuLV)-transformation of fetal liver and bone marrow and measure lambda5 and VpreB expression in these. Expression of lambda5, VpreB and mu H chains in normal cell populations will be analyzed by PCR (polymerase chain reaction) of cDNA and by the binding of anti-lambda and anti-mu-antibodies. Antibodies to lambda5 and VpreB peptides and the lambda5/VpreB/mu protein complex will be prepared. Transcriptional regulation will be measured by cloning discrete DNA fragments from the genomic clone of VpreB1 and lambda5 into CAT vectors and transfecting cell lines representing different stages of B cell differentiation. Expression of lambda5 and VpreB will be inhibited by anti-sense RNA and antibody in the cell lines derived above and the effect of this inhibition on pre-B cell-stromal adherence/stimulation and on subsequent H and L gene rearrangement will be determined.